The invention relates to derivatives of 25-hydroxy vitamin D, a synthesis thereof, and a method of measuring 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D in samples.
The D-vitamins or calciferols arise from their provitamins through a cleavage, catalysed by sunlight, of the B-ring in the sterane rings. Their most important representatives are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalci-ferol), which differ slightly only in the side chains, but whichxe2x80x94so far as knownxe2x80x94are similarly metabolised and have identical biological effects. Whereas provitamin D2 must be taken in with the food, the provitamin D3 can be formed in the human organism. So far as not more specifically designated by means of indices, the term vitamin D comprehends in the following in general all vitamin D forms. Vitamin D formed in the skin or taken in with food is bound in the plasma by vitamin D binding or transport proteins (DBP), transported to the liver and there metabolised to 25-hydroxy vitamin D (25-OH-D). The vitamin D binding protein DBP is also known as Gc-globulin or group specific component (J. G. Haddad in J. Steriod Biochem. Molec. Biol. (1995) 53, 579-582). Over 95% of the 25-hydroxy vitamin D measurable in the serum is as a rule 25-hydroxy vitamin D3. 25-Hydroxy vitamin D2 is only found in greater proportions if the person is receiving medication with vitamin D2 or, as is frequently the practice in the United States, foodstuffs are supplemented with vitamin D2.
25-Hydroxy vitamin D is the prevailing vitamin D metabolite in the blood circulation and its concentration in the serum generally indicates the vitamin D status, i.e. the extent to which vitamin D is available to the organism. If needed, 25-hydroxy vitamin D is metabolised in the kidneys to 1xcex1,25-dihydroxy vitamin D, a hormone-like substance with great biological activity. The determination of 1xcex1,25-dihydroxy vitamin D indicates how much vitamin D is present in the activated form.
The determination of 25-hydroxy vitamin D in a sample is preferably effected in accordance with the principle of competitive protein binding analysis, whereby on the basis of the displacement of radioactive 25-hydroxy vitamin D from the binding sites of a vitamin D binding protein, the 25-hydroxy vitamin D present in the sample can be quantified. Also, over the last several years, radioimmunoassays using 125I-labelled vitamin D derivatives and antibodies for vitamin D derivatives have established themselves in diagnosis. The data of the normal level of 25-hydroxy vitamin D in serum vary depending on the laboratory. It is, however, agreed that the concentration of 25-hydroxy vitamin D in the serum is as a rule greater than 5 ng/ml and smaller than 80 ng/ml. The competitive protein binding analysis requires the use of a radioactive vitamin D derivative which must have the same protein binding characteristics as 25-hydroxy vitamin D. The same applies also for the competitive binding analysis for the biologically active 1xcex1,25-dihydroxy vitamin D and other vitamin D metabolites.
European patent specifications 0 312 360 and 0 363 211, and Tanabe et al. in J. Chem. Soc., Chem. Commun. 1989, 1220-1221 and J. Nutri. Sci. Vitaminol., 1991, 37, 139-147, disclose various 125I-labelled hydroxy- and dihydroxy vitamin D derivatives and their use in binding studies. These derivatives suffer the disadvantages that they are problematic to produce and are extremely labile. Light, radioactive rays, protons, hydrogen, enzymes, free radicals or the presence of iodine in free or bound form have great effect on the configuration and the binding characteristics of the vitamin D derivatives to vitamin D binding protein DBP or specific antibodies. Above all, they can cause or catalyse a rotation of the A-ring in the sterane system. The 3xcex2-hydroxy-group of the vitamin D molecule is thereby rotated into the pseudo-1xcex1-position, so that 5,6-trans-vitamin D is obtained. The so-called pseudo-1xcex1-hydroxy-analogs of vitamin D may be metabolised similarly to vitamin D, but they have a structure which is different in significant points and are not bound or are significantly more poorly bound by vitamin D binding proteins such as for example DBP/Gc-Globulin or anti-vitamin D antibodies. 
The above-described re-arrangement is to be understood as an example. Other chemical reactions and re-arrangements also occur. The same applies for 3H- or 14C-labelled vitamin b derivatives. These vitamin D derivatives are likewise not so stable that they permit a reliable binding analysis. The radioactive marking additionally increases the costs of storage, transport and disposal and is generally disadvantageous for health and the environment. Further the half-life of 125iodine is relatively short. On the other hand, a competitive binding analysis with 3H- and 14C-labelled vitamin D derivatives requires particular scintillation counters and is more demanding in terms of equipment, with largely the same problems.
Ray et al., in Biochemistry, 1991, 30, 4809-4813 disclose the coupling of vitamin D3 with various colouring groups. The detection sensitivity for dye-labelled vitamin D3 derivatives is, however, too small that one might use them in a competitive binding analysis for natural vitamin D metabolites, apart from the fact that the dye-labelled derivatives are not stable in serum and further are particularly light-sensitive.
It is the object of the invention to make available vitamin D derivatives which can be employed in a competitive binding analysis or quite generally in immunoassays of vitamin D metabolites such as 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D. This presumes the following properties: first, that for the vitamin D derivatives, a detection sensitivity exists which is higher than, or lies in a lower range of concentrations than, the concentration of the sought after vitamin D metabolites in the samples; second, that the derivatives are stable in serum, plasma or urine under the usual protonic conditions and are stable with the respect to serum enzymes; and finally, third, that the derivatives are sufficiently stable with regard to light and storage, over weeks and months. This object is achieved by means of vitamin D derivatives having the formula 
wherein:
O represents the oxygen atom of an ether group;
X represents a substituted or non-substituted hydrocarbon group of 0.8 to 4.2 nm length, preferably a C8- to C12-group, which may have the usual heteroatoms such as S, O, N or P, most particularly preferred an hexamido-, octamido- or decamido-amidopropylether linker group;
Y represents hydrogen or a hydroxy group;
A a functional group which is bound with high affinity by a binding protein such as an antibody or vitamin D binding protein DBP;
R the side group of a vitamin D metabolite, preferably the side group of vitamin D2 or D3, particularly preferably the 25-hydroxylated side group of vitamin D2 or D3.
A high affinity is present when the dissociation constant (K) between the binding protein, e.g. the antibody or DBP, and the antigen or the functional group A is greater than 108. A dissociation constant greater than 1016 is advantageous for many applications. In a preferred embodiment A is selected from biotin, digoxigenin, tyrosine, substituted tyrosine, substituted amino acids, characteristic amino acid and peptide sequences, FITC, FITC-substituted tyrbsine, proteins and protein groups such as protein A and protein G or a further vitamin D derivative, most particularly preferred 25-hydroxy vitamin D.
The spacer group X is preferably selected from substituted and non-substituted C-bodies having a length of 0.8 to 4.2 nm, preferably about 0.12 nm. Particularly preferred is an amino carboxylic acid, in particular an amino undecanoic acid, peptide and keto group or a substituted or non-substituted amino polyether radical having a length of 0.8 to 4.2 nm, preferably about 0.9 to 1.5 nm. This spacing between the group A and the binding or detection site for the vitamin D radical is necessary so that the binding proteins can bind to the binding site concerned in each case and thereby do not interfere with one another. It is to be taken into consideration that for example for the vitamin D binding protein DBP (Gc-globulin) the 19-methylene group, if applicable the 1-hydroxy group of the A-ring and the vitamin D side chain belong to the recognition site and are received in a binding pocket. Similar applies also for specific antibodies against the various vitamin D derivatives. If the spacer group X is too short no further binding protein can bind to the selected functional group A along with the vitamin D binding protein. For the preferred example, this means that when the functional biotin group is located within the binding pocket of the vitamin D binding protein it is no longer accessible for the second binding protein, for example the streptavidin. On the other hand, if the spacer group X is too long, molecular folding can arise which likewise hinders the simultaneous binding of two binding partners.
Further, the spacer group in accordance with the invention surprisingly has a steric effect, since it clearly actively hinders a 180xc2x0 degree rotation of the A-ring. It is suspected, without being restricted to this theory, that the 3xcex2-oxygen atom of the ether group on the A-ring is hydrated corresponding to a natural hydroxy group and so prevents an attack on the 5,6-double bond, apart from other electronic and steric effects. A further important aspect is that the ether group in accordance with the invention cannot be dissociated by the esterases which are always present in serum or plasma.
Most particularly preferred is 25-hydroxy vitamin-D3-3xcex2-3xe2x80x2[6-N-(biotinyl)hexamido]amidopropylether of the formula II 
and the 1xcex1-hydroxy- and vitamin D2 analogs.
Further preferred are derivatives which contain as the second functional group a vitamin D radical. The advantage of these derivatives is that they contain no groups and compounds foreign to the system and so allow an increased sensitivity and reliability of the competitive binding analysis, also because they compensate, in a quantitative detection, for possible binding peculiarities of first and second binding of the vitamin D binding protein. Particularly preferred are compounds of the following formula III: 
wherein R, Y and X are defined as in formula I above. Thereby, symmetrical vitamin D derivatives are particularly favourable.
The 25-hydroxy- and 1xcex1,25-dihydroxy vitamin D derivatives in accordance with the invention are surprisingly stable with respect to light, storage and serum and allow in all competitive immune diagnostic methods a sensitive, reliable quantitative determination of vitamin D metabolites such as 25-hydroxy- and 1xcex1,25-dihydroxy vitamin D, for example for routine diagnostic use in human or veterinary medicine and in research.
In accordance with the invention the compound having formula I is obtained by means of a method including the steps: a) cyanoethylation of the 3-hydroxy group of vitamin D or 25-hydroxy vitamin D with acrylonitrile in a suitable solvent such as acetonitrile in the presence of potassium hydride and tertiary butanol; b) reduction of the resulting nitrile group with a mixture of lithium hydrid and lithium aluminium hydride to an amine; and c) linking a spacer group, if appropriate with a functional group A, to the amine, for example biotinylation of the compound with an active biotinylation reagent such as LC-BHNS or, to obtain a vitamin D derivative in accordance with formula III, coupling of two amino-vitamin D groups, by means of condensation, with a dicarboxylic acid such as sebacinic acid, by means of carbodiimide.
The method in accordance with the invention for the production of functional vitamin D derivatives gives higher yields with shorter reaction times. Different from conventional methods, there is effected namely in step a) the cyanoethylation of the 3-hydroxy group in the presence of potassium hydride and tertiary butanol. By this means it is achieved that cyanoethylation is effected only at the 3-hydroxy group of vitamin D and the other hydroxy groups of the vitamin D are protected from reaction. The reaction is effected at 0 to 20xc2x0 C., preferably at 5 to 8xc2x0 C. in a neutral solvent medium such as acetonitrile.
In the subsequent reduction, the nitrile group of the cyanoethylether is quantitatively reduced into the amine, which can then be relatively simply linked with another functional group, for example by means of reaction with a commercial available biotinylation reagent.
The invention includes additionally the use of the functional vitamin D derivatives in accordance with the invention in methods for detecting 25-hydroxy- and 1xcex1,25-dihydroxy vitamin D in serum, plasma, urine or another sample. Here, the functional vitamin D conjugate in accordance with the invention is employed either as an intermediate, whereby the vitamin D binding protein and native vitamin D metabolites compete for the binding site, or is employed itself as competitive binding component to native vitamin D. The quantitative detection method is preferably an EIA, ELISA, RIA, IRMA, LiA or ILMA, FIA or IFMA in test systems which are to be worked manually or in versions adapted to automatic testing machines, in liquid phase as well as solid phase technology.
A particularly preferred method for detecting 25-hydroxy- and 1xcex1,25-dihydroxy vitamin D derivatives include the steps: a) coating a carrier with streptavidin, b) addition of one or a plurality of a multifunctional biotin-vitamin D derivatives, c) addition of the sample and a defined quantity of vitamin D binding protein, d) detection of the bound binding protein with labelled anti-vitamin D binding protein antibodies. The labelling of the anti-vitamin D binding protein antibodies can be direct, for example a radioactive marking, or also indirect, for example by an enzyme or an active enzyme fragment such as peroxidase, which is capable of catalysing a colour reaction.
A further preferred method for detecting 25-hydroxy- and 1xcex1,25-dihydroxy vitamin D derivatives includes the steps: a) coating a carrier with anti-vitamin D binding protein antibodies, b) adding the vitamin D binding protein, c) adding the sample and a defined quantity of biotin-vitamin D derivative, d) detecting the quantity of bound derivative with labelled streptavidin. The streptavidin is preferably indirectly labelled with peroxidase; the carrier is preferably a reaction vial wall, for example of a microtitration plate, or particles of polymer or magnetic material or both, for example plastic or cellulose microparticles.
These methods make possible a non-radioactive quantitative detection of 25-hydroxy- and 1,25-dihydroxy vitamin D, without extensive safety measures being required. The competitive methods proposed here are thus suitable for routine investigations within in the scope of osteoporosis prophylaxis, in the case of a suspected D-hypovitaminosis or D-hypervitaminosis, for diagnostics in general, and in research.
A further aspect of the invention is a kit for detecting vitamin D metabolites such as 25-hydroxy- and 1,25-dihydroxy vitamin D, which inter alia contains the functional vitamin D derivative in accordance with the invention. The kit includes a vitamin D binding protein (Gc-globulin) which can be freely selected, anti-vitamin D binding protein antibodies, streptavidin and pre-prepared or non-pre-prepared microtitration plates and/or magnetic or other microparticles and other reagents.